The present invention relates to a probe which is useful for detecting and identifying the causative bacteria of infectious diseases, especially Streptococcus pneumoniae which is a representative causative bacterium of bacterial pneumonia.
In pathology, infection is defined as an invasion and an establishment of an anchorage for growth in a host organism by a pathogenic organism (hereinafter referred to as xe2x80x9cbacteriumxe2x80x9d). The outbreak of a disease caused by proliferation of a bacterium in vivo depends upon the interrelationship between the resistance of the host organism and the virulence of the bacterium.
To improve therapeutic systems for treatment of infectious diseases, especially inflammatory diseases caused by Streptococcus pneumoniae and the like in pulmonary lobes and bronchia, namely bacterial pneumonia, among the infectious diseases, has been urgent problem in the field of this art. Such bacterial pneumonia is triggered by an attack with bacteria, e.g., Streptococcus pneumoniae, Staphylococcus aureus and the like which cause inflammation predominantly in alveoli. When suffered from bacterial pneumonia, generally from clinical aspect, inflammatory symptoms in upper airway followed by sudden chill and shiver attach, then crisis of high fever around 40xc2x0 C., and terrible cough, stethalgia and sputum have appeared.
Further, the bacterial pneumonia is a serious and exigent infectious diseases in which severe systemic symptoms such as malaise, anorexia and headache have involved, presenting dyspnea, cyanosis, and could be accompanied by bacteremia, cerebrospinal meningitis or arthritis as the complication thereof, finally could sometimes lead to a lethal process.
Thus, improvement in rapid method for therapy of bacterial pneumonia has been awaited since appropriate therapy to be put into practice in early stage based on accurate and quick diagnosis is necessary.
Moreover, when suffered from an infectious disease including pneumonia, it has been believed that phagocytes including neutrophils, monocytes and macrophages primarily function in defense of the body, and that exuded bacteria from the texture of the phagocyte which had predominantly grown have appeared into blood.
In general, bacterial pneumonia is defined as a case wherein the ability of phagocytosis by cells cannot overcome the virulence of the bacteria and then the bacteria such as Streptococcus pneumoniae settle on the pulmonary lobe and the tissue of bronchia to cause inflammation. In conventional method for diagnosing bacterial pneumonia, the following items should be checked: 1) clinical symptoms; 2) culture of a specimen; 3) gram-staining of the bacteria contained in the specimen; and 4) shock state. After those items have been clarified, the course of therapy has been oriented. In its typical case, the above mentioned clinical symptoms, stethendoscopic findings, and increase in neutorophils, increase in acute phase response substances such as CRP (C-Reactive Protein) make speculation of diagnosis possible, however, for definitive diagnosis, the causative bacteria must be searched and determined from the specimen such as sputum, hydrothorax or blood, and then treatment must be conducted using proper antibiotics responding to the species of the bacteria. Accordingly, rapid and reliable identification of the causative bacteria has been awaited in the clinical site.
Additionally, novel types of pneumonia e.g., Legionellosis and Pneumocystis carinii pneumonia are identified, and resistant strains such as MRSA (methicillin-resistant Staphylococcus aureus) have been appeared recently, the importance in searching the causative bacteria have been growing.
However, as a matter of fact, difficulties have usually accompanied in confirming the causative bacteria. Especially, in case of community acquired pneumonia, it is known that therapy is initiated in 30-50% of the cases under such circumstances wherein the causative bacteria thereof are not clarified yet. As a method for identifying the causative bacteria in a patient who is suspected to be suffered from bacterial pneumonia, the following common procedures are adopted: employing the sample collected from sputum, secretion from upper airway, hydrothorax, topical focus, or blood as a specimen to estimate applicability of the sample as a test material by observing inflammatory cells of smear thereof, then, determining cell type by Gram staining e.g., gram negative or positive, and coccus or bacillus, and finally, culturing the bacteria using selection medium to identify the causative bacteria.
In accordance with this method, however, culturing the bacteria takes long time, and contamination of indigenous bacteria could not be avoided as well. Otherwise, in case that a lot of antibiotics have been administered when bacterial pneumonia had been suspected, even though bacteria are contained in the specimen, proliferation or growth would often be prevented, thus the rate of success in culturing the bacteria from the specimen has become actually quite low.
Although available subroutine methods including instrumental analysis method of constituents of bacteria and metabolic products by bacteria (See Yoshimi Benno, xe2x80x9cQuick identification of bacteria with gas chromatographyxe2x80x9d, Rinsho Kensa, Vol. 29, No.12, 1618-1623, November 1985, Igaku Shoin.), a method utilizing a specific antibody (See, Japanese Patent Provisional Publication No.60-224068.), and a hybridization method utilizing a specificity of DNA (Japanese Phase Patent Provisional Publication No. 61-502376) have been developed, any of which requires separation, culturing and growing of the bacteria.
On the other hand, as an established method based on the function of the phagocyte in infectious diseases, there is a method to examine a stained smear of buffy coat wherein leukocytes in the blood sample are concentrated, under an optical microscope. Generally speaking, the rate of detection of bacteria in buffy coat specimens from adult bacteremia patients is 30% at most, which is similar to that in blood specimens from an earlobe. However, it was reported that in case that the patients were newborn children, bacteria had been detected in seven cases in ten cases total (70%). Therefore, information concerning the presence of bacteria in peripheral blood obtained by a microscopic examination on a smear is an important index for therapy.
Since the above mentioned conventional methods necessitate the pretreatment which requires at least three to four days in total including one to two days for selective isolation of bacteria from a specimen, one day for culture, and one or more days for operation of fixation, and then the culture thereof is continued in practice until the bacteria grow. Therefore, in many cases, the pretreatment requires one week or more. Furthermore, there has been a risk on contamination of a other bacteria which could not be distinguished from the causative bacteria during the culture period.
As an important matter, under such circumstances above described, the number of bacteria that can be grown is small even under appropriate conditions for culture, because many bacteria in a specimen to be grown have been ingested into phagocyte, dead or on a static state due to antibiotics administered. Therefore, the actual detection rate of bacteria is as low as about 10% when the clinical specimen culture method is employed. In the other words, for the present, the presence of bacteria in 90% of the examined blood from the patient suspected clinically as suffering from pneumonia, which has been cultured for further one or more days, could not be proved after all.
Thus, in light of the situation above, the present practice depends on a trial and error treatment method, starting when pneumonia is clinically suspected without awaiting the detection results of the identification, wherein an antibiotic having the effectiveness for the widest range of the causative bacteria is administered first while the causative bacteria is still unknown, and if the antibiotic is not effective after one or two days, then another antibiotic will be tested, regardless of the fact that determination of the causative bacteria and selection of the suitable antibiotics are required.
According to the method to stain the bacteria in a specimen, the constituents of the living body are likewise stained together with bacteria, therefore, a skilled experience to identify bacteria quickly according to their image through a microscope is required, then there may be cases that can be hardly identified.
Although pneumonia is a disease wherein a rapid and exact diagnosis has been required as stated above, the conventional diagnosis method could not have satisfied such requirements.
The present invention was accomplished in view of the above-described problem in the art, and according to one aspect of this invention, there is provided a probe having a specific reactivity with DNA or RNA obtained from causative bacteria of the infectious diseases, especially, Streptococcus pneumoniae which is the representative causative bacteria of bacterial pneumonia, and nucleotide sequences of a portion of the gene essentially included in Streptococcus pneumoniae being elucidated.
Namely, the probe of the present invention allows significant detection of remaining bacterial DNA, the bacteria being incorporated into phagocytes and destroyed, thereby quick and accurate detection method of the causative bacteria of the infectious diseases would be available without culture and/or growth of the bacteria. Moreover, when a primer is designed with reference to information on the nucleotide sequence of the probe, causative bacteria can be identified without hybridization step, through amplifying DNA by means of a PCR technique.
Additionally, when a non-radioactive probe, e.g., a biotinylated probe, is used for hybridization step, detection in a general laboratory can be performed as well using an optical microscope, and the detection process will be carried out rapidly and simply.